The codon was optimized for the bovine nebovirus(BNeV)VP1 gene and the recombi-nant plasmid pFastBac-Dual-VP1 was constructed,and BNeV-VP1 virus-like particles(VLPs)were prepared using a baculovirus expression system,and identified by Western blot,indirect im-munofluorescence and electron microscopy.Successfully validated VLPs were mixed with MF59 adjuvant and CpG-ODG,and mice were immunised by intramuscular injection and evaluated for immunity effects.The results showed that the optimized CAI(codon adaptation index)of VP1 gene was 0.93 and the GC content was 60.4％.The constructed recombinant plasmid was trans-formed into DH10Bac for blue-white spot screening,and after successful verification,it was trans-fected into SF9 cells to obtain recombinant baculovirus Baculo-BNeV-VP1.BNeV virus-like parti-cles with diameters ranging from 35 to 40 nm were observed under the electron microscope,and both IFA and Western blot experiments proved that the target proteins were successfully ex-pressed and biologically active,and protein optimisation revealed that the highest protein expres-sion was found at the infectious dose MOI=0.5.Mice were immunized by intramuscular injection after 50 μg of VLPs were mixed with MF59 adjuvant and CpG-ODN.The results showed that the VLPs immunization group produced IgG antibodies 7 days after the first dose,and the antibody ti-ter increased gradually,reaching a maximum of 1∶102 400,and declined at 35 d,but still main-tained a high level;The blocking titer BT50 is up to 640,which can induce the production of BNeV VP1-specific blocking antibody in mice.In this study,the baculovirus expression system was used to express the VP1 protein of BNeV,and BNeV VLPs were successfully constructed,which could induce humoral immune response in mice,which provided a reference for the follow-up research of BNeV vaccine.

Codon optimization was performed for the M and HN genes of bovine parainfluenza virus type 3(BPIV3),and the recombinant shuttle plasmid Dual-M+HN was constructed.BPIV3 VLPs was prepared using the baculovirus expression system,and verified by Western blot,IFA and elec-tron microscopy.The successfully verified virus-like particle(VLPs)were mixed with MF59 adjuvant and CpG-ODN immunoenhancer to immunize mice by intramuscular-injection,and BPIV3 inactivated vaccine group and adjuvant control group were set up.The immune effect of BPIV3 VLPs was evaluated by monitoring mouse serum specific antibodies,neutralizing antibodies and hemagglutination inhibition antibodies.The results showed that the optimized codon adaptation in-dex(CAI)of the M and HN protein genes were 0.96 and 0.95,respectively,and the CG content reached 54.1％and 53.1％,respectively.The constructed recombinant plasmid was transformed in-to DHI0Bac for blue and white spot screening.The validated recombinant rod particles were trans-fected into Sf9 cells to obtain the rod-shaped virus pFastBac-M+HN.Under electron microscopy,BPIV3 VLPs with a diameter of approximately 180 nm were observed.IFA and Western blot ex-periments confirmed the successful expression and biological activity of the target protein.Through protein optimization,it was found that the protein expression was highest at an infection dose of MOI=5.After mixing 50 μg VLPs with MF59 adjuvant and CpG-ODN,mice were immunized by intramuscular injection.The results showed that the antibodies in the VLPs immunized group be-gan to rise at 2 weeks of the first immunization and reached their peak at 21 days of the second im-munization,with an average IgG antibody titer of 1∶40 228;The average titer of neutralizing anti-bodies is 1∶298;The titer of hemagglutination inhibition antibody is 1∶549,reaching the level of inactivated vaccine(P≥0.05),indicating that the VLPs prepared in this experiment can induce hu-moral immune response in the body.In summary,this study successfully prepared VLPs capable of self-assembly expression of BPIV3 HN and M proteins,and induced humoral immune response in mice,providing research basis for subsequent BPIV3 VLPs vaccine research.

The codon was optimized for the bovine nebovirus(BNeV)VP1 gene and the recombi-nant plasmid pFastBac-Dual-VP1 was constructed,and BNeV-VP1 virus-like particles(VLPs)were prepared using a baculovirus expression system,and identified by Western blot,indirect im-munofluorescence and electron microscopy.Successfully validated VLPs were mixed with MF59 adjuvant and CpG-ODG,and mice were immunised by intramuscular injection and evaluated for immunity effects.The results showed that the optimized CAI(codon adaptation index)of VP1 gene was 0.93 and the GC content was 60.4％.The constructed recombinant plasmid was trans-formed into DH10Bac for blue-white spot screening,and after successful verification,it was trans-fected into SF9 cells to obtain recombinant baculovirus Baculo-BNeV-VP1.BNeV virus-like parti-cles with diameters ranging from 35 to 40 nm were observed under the electron microscope,and both IFA and Western blot experiments proved that the target proteins were successfully ex-pressed and biologically active,and protein optimisation revealed that the highest protein expres-sion was found at the infectious dose MOI=0.5.Mice were immunized by intramuscular injection after 50 μg of VLPs were mixed with MF59 adjuvant and CpG-ODN.The results showed that the VLPs immunization group produced IgG antibodies 7 days after the first dose,and the antibody ti-ter increased gradually,reaching a maximum of 1∶102 400,and declined at 35 d,but still main-tained a high level;The blocking titer BT50 is up to 640,which can induce the production of BNeV VP1-specific blocking antibody in mice.In this study,the baculovirus expression system was used to express the VP1 protein of BNeV,and BNeV VLPs were successfully constructed,which could induce humoral immune response in mice,which provided a reference for the follow-up research of BNeV vaccine.

Codon optimization was performed for the M and HN genes of bovine parainfluenza virus type 3(BPIV3),and the recombinant shuttle plasmid Dual-M+HN was constructed.BPIV3 VLPs was prepared using the baculovirus expression system,and verified by Western blot,IFA and elec-tron microscopy.The successfully verified virus-like particle(VLPs)were mixed with MF59 adjuvant and CpG-ODN immunoenhancer to immunize mice by intramuscular-injection,and BPIV3 inactivated vaccine group and adjuvant control group were set up.The immune effect of BPIV3 VLPs was evaluated by monitoring mouse serum specific antibodies,neutralizing antibodies and hemagglutination inhibition antibodies.The results showed that the optimized codon adaptation in-dex(CAI)of the M and HN protein genes were 0.96 and 0.95,respectively,and the CG content reached 54.1％and 53.1％,respectively.The constructed recombinant plasmid was transformed in-to DHI0Bac for blue and white spot screening.The validated recombinant rod particles were trans-fected into Sf9 cells to obtain the rod-shaped virus pFastBac-M+HN.Under electron microscopy,BPIV3 VLPs with a diameter of approximately 180 nm were observed.IFA and Western blot ex-periments confirmed the successful expression and biological activity of the target protein.Through protein optimization,it was found that the protein expression was highest at an infection dose of MOI=5.After mixing 50 μg VLPs with MF59 adjuvant and CpG-ODN,mice were immunized by intramuscular injection.The results showed that the antibodies in the VLPs immunized group be-gan to rise at 2 weeks of the first immunization and reached their peak at 21 days of the second im-munization,with an average IgG antibody titer of 1∶40 228;The average titer of neutralizing anti-bodies is 1∶298;The titer of hemagglutination inhibition antibody is 1∶549,reaching the level of inactivated vaccine(P≥0.05),indicating that the VLPs prepared in this experiment can induce hu-moral immune response in the body.In summary,this study successfully prepared VLPs capable of self-assembly expression of BPIV3 HN and M proteins,and induced humoral immune response in mice,providing research basis for subsequent BPIV3 VLPs vaccine research.